RESUMO
Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.
Assuntos
Trifosfato de Adenosina/metabolismo , Cílios/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucose/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cerebelo/citologia , Cerebelo/metabolismo , Cílios/ultraestrutura , Creatina Quinase Forma BB/genética , Creatina Quinase Forma BB/metabolismo , Expressão Gênica , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glucose-6-Fosfatase/genética , Glicólise , Masculino , Microssomos/metabolismo , Microssomos/ultraestrutura , Neurônios Receptores Olfatórios/citologia , Fosforilação Oxidativa , Ratos , Ratos Sprague-Dawley , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Sequência de Aminoácidos , Animais , Anoctaminas/metabolismo , Canais de Cloreto/genética , Cílios/metabolismo , Clonagem Molecular , Células HEK293 , Humanos , Íons/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Alinhamento de Sequência , TransfecçãoRESUMO
BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.